All the responses about PCR-inhibition and trying other primers are reasonable, except that you state that the raw genomic DNA for this particular sample does not show a band in the gel either... suggesting that this is not something to do with the PCR, but rather with the DNA.

1) As Zhi said, pure water is not really the best resuspension medium; with no buffering capacity, it is easy for the pH to shift dramatically enough to cause the DNA to degrade. A better medium is a weak Tris-EDTA buffer (e.g., 10 mM Tris, 0.05 mM EDTA) which will preserve the pH and inactivate DNAses (by capturing free Mg) while not interfering with most downstream applications.

2) Also, the nanodrop can easily give the false impression that DNA is present in your extraction because many things can absorb in the 260/280 wavelengths; you at least need to carefully examine the UV profile to ensure that it follows the normal pattern for a clean DNA sample. Better still, if you're looking for accurate quantitation, you should use a technique that measures only dsDNA (such as a Qubit or a Bioanalyzer, both of which have their pros & cons)... a cheap alternative for a crude estimation is to run a serial dilution of the sample in a gel, and compare it to a quantitative ladder... or see what dilution is required to make the band shift from being extremely faint to vanishing in a clean gel... some detective work on your part with some known DNA concentrations will tell you the approximate concentration of such a sample.

You can try diluting your template, as too much can inhibit PCR. If that doesn't work, try alternative 16S primers. "Universal" primers sometimes aren't (we've seen this in our research).

Hi,

I once had encountered such a problem and I might speculate that by a rare probability if in that particular bacterial clone there has been a point mutation de novo and its in the region of your interest i.e at the primer binding site (either forward or reverse), then it happens since sometime in the initial bases or the kind of base mutated affects the binding of the primer to template.

If you really want to know the reason, you might want to design another forward keeping the reverse same or vice versa and see if you get a band. My observation with such a thing showed that I had a single base change which was making my reverse primer inefficient in binding and hence no signal. You can simply check the effectiveness of your DNA by taking any other primer set for some other gene if you have and amplify.

Hi Jennifer,

Based on your describing, I think there are several possibilities: 1) is the DNA degraded? Pure water is not a perfect buffer to keep DNA. Did you run the 7th genomic DNA on a gel? 2) Did the PCR system work well? If you have a positive control at the same time, you will know it. 3) The target region of the amplification (16s rRNA) may be covered by higher structure of DNA. Extending the pre-denature time to 10min probably will help you.

I think you have too much template. Try to use the 1:10 diluition of your 7th as template in the PCR.

Some bacteria produce a lot of carbohydrates that inhibit PCR. Try diluting, you should be able to dilute to about 50 pg/ul and still get amplification with 16S primers.

I've just 2 question for you:

1. Are the 7th sample inherit with the other 6?

2. If not, then why do you think the PCR will amplify the fragment without the proper primer?

If the 7th sample is also part of the same family, than probably you have "problem" with the primer (if the repeats give the same results). => you found something interesting.

As some colleagues already pointed, it looks like you have a) too much DNA and it inhibits the PCR reaction, b) some internal inhibitor is blocking the reaction. Just an advice, don't trust too much the Nanodrop measure. It is good for know "yes" or "not" but it does not use to match with more accurate DNA measure methods if the amount of DNA is not big.

try doing a colony PCR (same PCR protocol but add a 'bit' of the colony instead of extracted DNA) - it wont give you the best sequence results but might be a good way to rule out some issues. (if you get a result with a descent sequence, you might not even need to troubleshoot further!) the PCR gods work in mysterious ways :).

Maybe you have a problem with your primers!

All the responses about PCR-inhibition and trying other primers are reasonable, except that you state that the raw genomic DNA for this particular sample does not show a band in the gel either... suggesting that this is not something to do with the PCR, but rather with the DNA.

1) As Zhi said, pure water is not really the best resuspension medium; with no buffering capacity, it is easy for the pH to shift dramatically enough to cause the DNA to degrade. A better medium is a weak Tris-EDTA buffer (e.g., 10 mM Tris, 0.05 mM EDTA) which will preserve the pH and inactivate DNAses (by capturing free Mg) while not interfering with most downstream applications.

2) Also, the nanodrop can easily give the false impression that DNA is present in your extraction because many things can absorb in the 260/280 wavelengths; you at least need to carefully examine the UV profile to ensure that it follows the normal pattern for a clean DNA sample. Better still, if you're looking for accurate quantitation, you should use a technique that measures only dsDNA (such as a Qubit or a Bioanalyzer, both of which have their pros & cons)... a cheap alternative for a crude estimation is to run a serial dilution of the sample in a gel, and compare it to a quantitative ladder... or see what dilution is required to make the band shift from being extremely faint to vanishing in a clean gel... some detective work on your part with some known DNA concentrations will tell you the approximate concentration of such a sample.

try using Chelex 100 to extract your DNA. It's a contamination problem of some kind.

Did you repeat the pcr using the 7th dna??

Protein and RNA removed (proteinase K and RNase enzymes included in kit), as indicated by A260/280 ratio = 1.8-2.0.

No trace organics, as indicated by A260/230 ratios >1.7.

Either way - maybe try less template in PCR - try 1/10 and 1/100 dilutions of template with water before 16S.

BTW - what's different with #7 - is it Strep? with autolysis properties? Maybe you have degradation before extraction?

If you don't see any DNA on number 7 with a control agarose gel, then it's not that surprising not to get amplification of any gene. there are several possible explanations not to get DNA when using a kit. But to be honest I would not waste too much time and simply reuse the same culture N7 to make another DNA prep (takes 10min) then do another PCR.

My understanding is that you ran the extracted material (non amplified, as it comes out of the extraction procedure) on the gel and in 6 out of the 7 cases you see a band, in one case you don't see a band but you have a nice absorbance, and a white pellet. If my understanding is correct it seems that you have a problem of degradation, for that case, during or after the extraction. Nonetheless, you could still have a nice absorbance and pellet from the free nucleotides. If I were you I would re-do the extraction for that particular case, just to make sure you did not make any mistake during the procedure.

You could tried a DNA extraction withouth a kit, eg phenol based method and after visualized the DNA tried again the pcr...

Possibly as you suggest, there is too much DNA.

This bacterial strain belongs to a particular group?, there are some types in which the DNA extraction is realized with many pollutants. These Gram-positive bacteria, perhaps it is better to DNA extraction with phenol-chloroform. Or you could dilute and go through some column to purify this material better.

excuse my English, SUERTE

Hi Jennifer, it sounds like an inhibition by excess of template!

It occurs often when you have more template than primers

Did you try to dilute? I suggest to dilute the sample and use no more than 5ng of it in the pcr reaction. I also suggest the use of DNase/Rnase free water just in case.

Finally if it still does not work there are 2 other possibilities: First an inhibitor of polymerase is present in your purified DNA (and usually in this case diluting is enough), or your DNA is degraded (you can verify it using a bioanalyzer, but you should at least see smears on your gel)

Hope it will help you

Good luke

Dear

in my opinian it's better you try anotherr way to DNA extraction,if you don't have band in PCR check your primmer to be specific.

Jennifer, you may have a strain-specific DNase. I had this problem with some isolates of mycobacteria many years ago. I recommend that you try boiling the bacteria for 10 minutes before the extraction to denature (most) enzymes, or using a guanidinium and/or phenol-based extraction method.

As most have already stated: no band on a gel - there is no DNA of any significant length. That can be a problem with the bacteria (nuclease activity degrading your preparation) or the column of the kit not working for example and you have just protein in there. Since the 260/280 looks ok, I think you have degradation (usually having a lot of nucleotides brings the 260/280 up very much, so a normal 260/280 would indicate degradation plus lots of protein probably.

I'm not a believer of the TE theory for elution. Talked with people who run a company that commercially produces plasmids in GMP years back and they said it is pretty simple: DNA has such a strong charge that no TE or whatever buffer in the world will be able to even slightly buffer THAT. So whatever the pH of your water might be before adding DNA to the mix ... as long as it is not full of Mg2+ ions, TE probably doesn't do a thing. While on the other hand TE can interfere with downstream applications like PCR's that do rely on Mg2+ for activity.

you should repeat the DNA extraction with the same kit and if the problem remain persist then run the PCR with increase template concentration. i think it will work.

I tend to agree with all that's been offered above. There is the remote possibility that what you saw for pellet following column elution was heavy on salts along with DNA, if you just didn't get a good wash through that column for some reason. That salt alone may be sufficient to largely inhibit your PCR. A couple of things I'd try before throwing the baby (DNA) out with the bathwater: 1)try running your amplification again, this time running it for 5-8 more cycles than your protocol calls for...if you have low but amplifiable DNA in there, that may give you a band; 2)try doing a second round of ethanol precipitation/pelleting/resuspension on the sample before you toss it...that should help you to wash out any inhibitors...then run the PCR again.

Hi: If you can't find the genomic DNA in the gel, Two thing may be happened. 1. your sample already degraded to smaller fragment . when you see it, it has run out of gel.so you have to short the time you run a gel and take look the band. If the DNA degraded, you still can get good concentration and 260/280 and 260/230 data. but you can fail to run PCR.

2. If you can sure that DNA is good and PCR positive control negtive control was perfectly come out. you can think about the sample DNA 16r GENE may be deleted.

Jennifer, all of the above are ways of probing, as from my opinion, I think the problem might not be with the PCR but the DNA quality itself. Check through this first and rerun and you will be sure to get result

I just want to tell you a couple of thing that already did not see in the other comments. Fisrt, be careful with DNases, Second, I suppose you blanked correctly the neno Drop, Third, if you can run a little of your DNA sample (need to be >10ng total) in a agarose gel (not forget the staining) you can see if you have possible bands. Fourth, be sure before nothing you have DNA, the amplification of 16SrRNA gene is so high the most of the time, so this is a clue that you need to check first your isolated product.

I supposes you did not repeat the extraction? or did you?

I would go for another round of extraction before jumping to any conclusion..!

There are lots of things which needs to be cared about..!

Hi Jennifer, among all the suggestions you got I would like to put the possibility of base changes at your primer annealing region. As Scott mentioned, you could try to use other primers. Try degenerated primers sequences or redesign your primers a lit bit up and downstream.

First question: Have you tried again to amplify it?

-Sometime a reaction just doesn't work without any reason....

Else there is many reason... Too small DNA quantity, too big DNA quantity, wrong primer for specific organism....

Reduce sample concentration

Hi. You do not mention the name of the species that you are having difficulty in isolating total DNA from. I suspect that (a) you may be getting incomplete lysis of the cells and (b) nucelase contamination may be degrading the DNA. The white pellet may be a combination of degraded genomic DNA and RNA.

I have previously had similar problems with Gram-positive bacteria. I found that methods that produced high yields of high quality genomic DNA from Gram-negative bacteria did not work with Gram-positive bacteria.

I suggest the following: After harvesting cells from a 20 ml culture very turbid broth culture resuspend cells in 2 ml of 0.1 M TRIS-EDTA buffer and at 10 microlitres of Diethylcarbonate (a nuclease inhibitor) (Sigma Chemical Co. is one source). Mix. Incubate on ice for 5 minutes. Centrifuge and discard the supernatant into an appropriate chemical waste container and resuspend in 0.2 ml of TRIS-EDTA. Add 2 ml of ice cold acetone. Mix by shaking for 30 seconds, allow to stand for 10 minutes on ice. Centrifuge again and discard the supernatant again into an appropriate chemical waste container. Air dry the pellet at 55 oC in a fume cupbooard. Resuspend the pellet in 2 ml of Tris EDTA and proceed with the use of the kit you were using to isolate the DNA. Be sure to follow the neccessary safety instructions when handling dietylcarbonate and acetone and consult your Institutes/University safety procedures for the handling and disposal of these wastes.

The pretreatments should eliminate any nuclease issue and think the acetoone strips capsular material from the cell walls making them accessible to enzymatic degradation of envelope proteins and chemical disruption of the membrane.

I find it works well with Gram-positives like Clostridium species in particular.

An alternative is to PCR directly from the culture. I'll see if I can find a method for that and send it on.

Hope it works out for you.

This method will work for you and it is very simple.

50ul of turbid broth containing the organism is placed on clean, sterile microscope slide. The drop is heat fixed to the slide by gently passing through a blue bunsen flame using a sterile steel tweezer (forceps) ten to twenty times for 2 seconds each time. (Similar to the heat fixing method used for Gram-strain prep).

Allow the samples to cool and then place 50ul of 1X PCR buffer onto the fixed cells and pipette up and down. Transfer 5ul of the sample into the PCR reaction vessel and carry out the PCR amplification. This takes less than 5 minutes and eliminate nuclease problems (by heat inhibition) and allows DNA to dissolve in PCR buffer directly by heat disruption of the envelope.

Give it a try and see how you get on. I am sure it will work.

As you said your DNA concentration after dilution in water was this is a pretty high concentration if you want to amplify DNA, Additionally you can try hydrated t it in a buffer solution as TRIS EDTA 10mM that is a good buffer to reduce degradation.

Also try to modify some concentrations of MgCl2 in your PCR increasing it can help you to decrease taq inhibition.

But at firsts I would dilute the DNA to reduce its concentration.

Good luck

Maybe you miss something in the 7th.

As you said your DNA concentration should be check, try with negative control PCR for you primer,

Jennifer, I also observe the same type of pellet sometimes. it dissolves readily but no dna band is observed. Few things I have been able to point out in my results. First, there is no intact DNA, second the pellet is either of degraded DNA or RNA contamination (sometimes even after effective rnase treatment), third there is some protein residue...Also i have seen many times that although Nanodrop is good but its reading are not always matching to ur gel performance. I would suggest you use Qubit Flurometer if u have for calculation, if u find ur nanodrop result unsatisfactory. TheNanodrop reading of 7th sample may suggest presence of something but if the A260/260 is between 1.75-1.85 only then u can be sure of RNA free DNA. I have been facing these problems for sometime. like 7 th sample many times i didnt got any amplification in PCR. I tried using many kits for that. but i found a motdified CTAB method published in 2010 to be working far better than any kit. it is a bit lengthy but gives good quality and quantity of dna. i suggest you try it once for 7th sample before troubleshooting with your PCR conditions.

you can find the here. Its for fungi but works for almost any cell pellet. I have used it on bacterial, animal cells and mitochondria also. it gives good result with slight modifications..

http://idosi.org/gjms/gjms5(1)/6.pdf

Good luck..!

Before ascertaining to a reason, please repeat the protocol as you have followed previously.

I would suggest you to dilute youre sample 5 to 10 times, perhaps the DNA concentration is too high for amplification. If you have qpcr you can try the amplification of the diluted and the concentrated samples and than see what the CT is and this gives you indication of your DNA concentration.

If this doesn't work I would think that the primer doesn't fit. Because the other samples give good amplification results I would not think that DNA degredation happened in your 7th sample and did not happen in all the other six. I would suggest to reduce the annealing temperature even make gradient PCR to find the best annealing temperature for this sample. clonning and sequencing can be done afterwards for making sure you get the right fragment.

hello,

complete failure of any amplification (even smears) may come, as stated by several above comments, by inhibitors or changes in the sequences binding the primers. I agree with those saying it's good to diminish dna amount since it also lowers inhibitors concentration.

If lack of binding occurs, it may be good to lower temperature and/or magnesium concentration or increase annealing time. But since this will lower specificity, this must be done with very low amounts of dna. But if you get smears or multiple bands, this also means that at least your polymerase is working.

You can also add in your reaction mixture a tad of a dna that you know is working well or a small aliquot of a previous amplification that worked . Failure of amplification will sign the presence of inhibitors

The fact that you can't see your actual DNA sample on a gel suggests that nothing is wrong with the PCR as it has worked for the other samples. I would suspect that something has gone wrong with the extraction. I would go back to step 1 and re-isolate the DNA from that sample. It may have been that the eppendorf you used may have had some dirt in it to start with.

A possibile reason can be contamination of your DNA fraction with DNAse which digests the fragments during amplification.

If you want to be sure for the presence and the quality of our DNA make a control PCR for your sample with Beta-tubulin primers. Once u get a 340 Bp band ull be sure that there is no prob with the DNA but maybe in ur specific primers, or in ur PCR conditions and concentrations.

I agree that the problem is the DNA not the PCR. Best is to re-isolate your DNA

How about your pipetting? In my case, I experienced like your case. It might be one more consideration for your problem.

I agree with Andre, you must be sure for the quality of your DNA sample. Also, DNA concentration is very important factors in PCR reaction, so, it is must be adjusted. Finally, modify PCR reaction conditions to get PCR products. Good luck

Maybe this sample's DNA extract has PCR inhibiting agents. Try re-isolating g DNA.

Vary and optimize the template conc for PCR. Also another source of error might be handling error.

The problem is the DNA not the PCR reaction. I agree with Massimiliano, Irina and Miloslav. Maybe you can try with a set of diluted samples to reduce the concentration of both DNA and inhibitors (10x, 100x..) and include replicates in your reaction to discard pipetting errors. If it is not working maybe try a new DNA extraction. Good luck

Use buffer TE for DNA elution and dilution DNA for PCR

If all reagents ok make 2nd pcr to increase the amount of pcr product which can detect by agarose electrophoresis

i have had the same problem.....I solved it by re-extracting DNA.

Reduce your DNA concentration set it in between 50 ng/ul to 100 ng/ul you will get amplification high concentration of DNA reduces primer attachment with DNA

Maybe you could try to load your DNA in a agarose gel in order to see if it's degraded or not. If you observe a smear, then your DNA is degraded and could explain your result. Good luck!

I notice you are amplifying 16s. Do you know the primers you are using will amplify the gene from the bug you are looking at? I know that the 16s primers I'm using will NOT pick up every single possible bacterial species. Do some in silico checks and maybe try and different primer set?

Try a dilution series for your sample to get rid of PCR inhibitors. I am doing DNA isolation from soil and activated sludge and always have a lot of inhibitors (humic acids and other) to get rid of. Start with a 1:10 dilution of your DNA and dilute up to 1:10.000. If this is still not working run a temparture gradient PCR to vary the annealing temp. If still not working you could also try another, more stable polymerase (I could give you the contact for a stable one if you need; just contact me).

Hope this works for you.

as mentioned before, alcohol contamination in your probe may be a problem and result in inefficient loading of your DNA probe for gel electrophoresis. In addition alcohol (and other contaminations) may inactivate yor PCR polymerisation resulting in absent products.

Furthermore, intact and degraded DNA (e.g. by DNAses,...) show very similar UV absorbance as well as ratios when quantified in a spectrometer. Therefore, quantifiable detection with a nanodrop is no adequate criterion for suitable DNA quality.

Repeat your preparartion of your 7th probe. Good luck

I had the same issue two years ago with one of my PhD student. I would say that the universal primers that are used did not pick up any gene for the 7th isolate so I would design another set of universal primers that might work. it is highly recommended to use different sets of universal primers . I had this issue before when Isolating unique unknown isolates.

The key thing is the agarose gel. If you do not see DNA, it either degrades rapidly or absent. White pellet might represent RNA and nanodrop can be not calibrated well. If bacteria you was isolating were similar, just do it again and try to dissolve the pellet in TE with 0.1mM EDTA. Good luck

You should reduce sample concentration, or may be DNA quantity is too little. Use nuclease free water or TE buffer and use negative & positive control for the detection of PCR accuracy.Good Luck.

I agree to everybody suggesting a different set of primers. One more, are you sure that this seventh isolate is bacterial and not fungal?

I presume you have carried out the tests many times and get the same result?

Suggestions: Redo experiment from start and obtain a new isolates. But keep original isolates for reference.

Possible causes on repeat results: 16s rRNA is a unique mutation; reasons given above; Aliens!!! :)

I suggest you to design another set of universal primers for your seventh isolate of bacteria. Probally have a ocurred a mutation in this gene sequence.Good luck!

If you still have your bacterial plate of the 7th isolate, try to pick the colony, inoculate into 20ul of distilled water and boil for 5-10 mins. Then, spin the tubes at 10,000 xg for 5 min. You can try use the crude DNA for you amplification to check whether if your primer cannot amplify the 16S of the 7th isolate.

If you get any band, that means that something went wrong during the extraction. I'm not sure how the kit from Epicentre works (does it use column base?). If it is column base, perhaps the DNA may stuck to the column. Or else, perhaps the lysis of the bacteria is not proper or can be due to other thing. All the best!

Actinomycetes are hard to lyse....so it may be actinomycete datz y u r not getting proper band....all the best.....

I would recommend two things to resolve this, in tandem. Firstly, although your DNA quantification and quality seems okay, I'd be very tempted to do a dilution of your template, in addition to the undiluted.

Secondly, and perhaps most importantly, it seems very likely that the primers that you're using are the problem. Simply try different ones and I'm sure you'll have success. It is likely that your 7th isolate has too many sequence mismatches to be of use.

May be this isolate degraded or contaminated

By the way, what is this 230/280 ratio? we never did that. Instead, we did 260/230 ratio.

before a concentrated precipitate genomic DNA with 0.1 vol of 3 M sodium acetate and 2 vol of ethanol for 2 hours and the resuspendes in TE.

perhaps try another one/combination of the 16S universal primers? perhaps the region of this bacterium targeted by the primers you are using has polymorphisms preventing annealing of the primer/s. The worrying thing is that you are not getting a genomic band from running the same on a gel raw... You have good concentrations but no band so there is no use doing a re-extraction - I would pick and grow a few more colonies from your cultures and see what you get! Good luck!

I probably think DNA is OK. So, there are some problems in PCR protocol, not in DNA extraction. Maybe it's primers set. As other researchers say. Good luck.

I agree with Laura Kelly. Too much template DNA in the reaction could possibly inhibit the reaction. Dilute your template DNA (1:10 or 1:100) and try again with same primers. If it does not work, pick another set of primers for 16s rRNA gene.

It is better you dilute the DNA by 1: 10 or 1:100 times and try PCR. Also perform hot start PCR. First denature DNA and primer by heating at 95C and snap chill them. Later on add other components of PCR. Also use sterile HPLC grade water or MilliQ water for dissolving DNA. I will suggest it is best to use 0.001M Tris buffer or 0.01M tris buffer for dissolving DNA.

In case of DNA not visible in agarose please go for UV spectrophotometric analysis or run native page if DNA is damaged it can be detected easily

Hi dear jennifer, maybe your DNA sample is broken, or your DNA is too long, and you need to more than two primer. and please check the PCR conditions again because maybe for example your DNA concentration is high.

We solved this problem. For anyone that is still interested. The results on the nano-drop indicating that we had plenty of high-quality DNA was not accurate. We discovered we were not getting cell lysis and that this was one tough bug. Later after switching to a bead-beating method instead of a chemical method, we were able to see strong genomic bands on the gel and amplify DNA using other genes than 16S. Our 16S primer set never did work.

I am glad you solved your problem! Note that when you use the nanodrop (or any spec), you should also inspect the overall shape of the spectrum. If you have a nice peak at 260 that looks like a bell curve/mountain sort of thing, that is best.

If you have a lot of salt in your sample, it will make a lovely pellet (in fact, a lot of any precipitation pellet is usually salt). The salt peak will be highest at the low end of the wavelengths - like 220-230 right where you start reading and can give signal at 260 and even 280 (but less at 280 as it is rapidly dropping).

If you have a giant salt peak with a hump at 260, then you know you have a salty sample, but there is some DNA (or RNA) there.

If you have a giant salt peak that doesn't even have a little bump around 260, it is possible that you only have salt or atleast you can't tell how much DNA you have.

Desalt your sample (reprecipitate and do two 70% ethanol washes or use a desalting column) and try again if salt will interfere with your next assay or you want to confirm whether or not you have DNA/RNA. Alternatively, just go into your next experiment knowing that there may be a lack of DNA.

First you run your genomic DNA samples on 1%Agarose gel. If you get a band that means its good. it happens some times during isolation of DNA it becomes sheared. Although it give absorbance in nanospectrophotometer but that is useless for PCR and other works. Even the sheared form of DNA gives higher absobance than normal one.

Dear,

I suggest to try amplify the 16s rRNA gene with PCR , please check the PCR conditions again, and run your genomic DNA samples on 1% Agarose gel.

The genomic DNA template may be diluted,2.More DNA polymerase can be added,3.Additives to the PCR reaction like Bovine Serum Albumin or Betaine will prevent or minimize PCR inhibitors.,4.NaOH t reatment of DNA neutralises inhibitors of Taq polymerase,5.Separation step with Centricon 100 or Microcon 100 filters can be tried.

Sounds like this problem was solved, two lingering questions though: was it a gram-positive bacterium with a tough pedtidoglycan outer-layer that caused the original problem with DNA extraction then? Since the original primers for 16S in this bacterium 'never' worked for you - does that mean your new set of primers now also work on the original extracts in retrospect? A certain ASF strain of gram- positive had the same problem here - and was solved the same way, except that the original primer set worked once lysis by bead method proved successful.

As a general principle, whether it is RNA or DNA, you should always test neat and a 1:10 dilution of your newly extracted nucleic acid, since this informs you empirically about possible inhibitors.

Since your 16s olignos never worked with this particular culture: have you thought of a mutation within the primer annealing site?

Regards

Lett Appl Microbiol. 1993 Feb;16(2):59-61.

PCR products generated from unpurified Salmonella DNA are degraded by thermostable nuclease activity.

Gibson JR, McKee RA.

SourceAFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, UK.

Abstract

Oligonucleotide primers designed from repetitive extragenic palindromic (REP) sequences were used to PCR-amplify Salmonella DNA. Unpurified template DNA, present in crude cell extracts, yielded an essentially identical banding pattern to that arising from the use of purified DNA. However, the PCR product derived from the crude preparations did not survive storage at 4 degrees C. This post-PCR DNA degradation, attributed to endogenous Salmonella nucleases, was inhibited by the addition of EDTA, or storage at -20 degrees C.

Does the quality of DNA reduces with number of PCR amplifications??

My suggestion is that, some time nano drop give good result but our DNA is fragmented so make sure DNA fragmentation with techniques as agarose gel electrophoresis and second thing try with changed primers and Taq pol.

I suggest that your DNA is good but the main problem may arise from 1) PCR conditions and annealing Temp, 2) the used DNA polymerase activity will affected by storage conditions and repeat uses 3) primers quality

Probably your isolate could be a yeast contamination with your sample

What about DNases, what about the concentration of the template and primers in your PCR reaction, what about the PCR conditions: temperatures and cycles, what about your primers?

Hi,

Sainz,why are you suggesting that it is yeast contamination? If that is the case,how could the samples be rescued and get the required specific DNA band?

Thank you

I dont know whether you did the same procedure twice or not. It can be a hand made error (that is quite normal). If same thing happened in the repeated work then you have to go for the protocol.

First of all, you must state reaction control in order to discriminate some abnormalities in PCR. If you look degradation in genomic agarose gel 0.8%, then reviewed again all reactives used in extraction procedures, perhaps nick band was just in a place intermediate where primers flanked.

Besides you could try to amplify anothen gen fragment to evaluated a little more the genome integrity.